1. Field of the Invention
The invention concerns methods for the electrochemical determination of factor Xa inhibitors, especially of heparins and heparin derivatives as well as direct factor Xa inhibitors in blood samples.
In addition the invention concerns test elements based on dry chemistry and test element analytical systems for the electrochemical determination of factor Xa inhibitors, especially of heparins and heparin derivatives as well as direct factor Xa inhibitors in blood samples.
2. Description of Related Art
Anticoagulants which in particular also include heparins are often used in clinical practice for the prophylaxis and therapy of haemostatic disorders (disorders of the coagulation system). Heparins and heparin derivatives are used especially for the therapy and prophylaxis of thromboembolic diseases. They are very effective in the prophylaxis and treatment of leg vein thromboses, pulmonary embolisms and for treating unstable angina pectoris and acute myocardial infarction. They are also often used during operations and in particular for cardiological procedures (bypass) and for blood transfusions.
The action of heparins is mainly based on their interaction with antithrombin III (ATIII) as a result of which they change the conformation of ATIII. This accelerates the inactivation of certain coagulation enzymes (thrombin (FIIa), factor Xa (FXa) and factor IXa) and thus coagulation is prolongated. Heparins can thus be classed as factor Xa inhibitors. Other factor Xa inhibitors are for example certain oligo-saccharides such as the pentasaccharide Fondaparinux or low-molecular-weight direct factor Xa inhibitors which are still in clinical development and can be assigned to different classes of substance. In addition to unfractionated heparins (UFH) which have been used for a long time, various fractionated heparins or low-molecular-weight heparins (LMWH) have been used since the end of the 1980s. Fractionated heparins have now replaced the UFHs for many indications and are prepared from unfractionated heparins by chemical or enzymatic depolymerization to form fragments which have only about ⅓ of the size of the standard heparin. This weakens among others the effect of these LMWHs on thrombin whereas factor Xa is preferentially inactivated. Fractionated heparins have other advantages over conventional unfractionated heparins as a result of their more advantageous pharmacokinetics. A review of these classes of substance with regard to their clinical action and significance may be found in “Heparin and Low-Molecular-Weight Heparin” (Mechanisms of Action, Pharmakokinetics, Dosing, Monitoring, Efficacy and Safety), Hirsh et al.; Chest 2001; 119:64p-94p.
Patients which have been administered unfractionated heparin are required to be monitored due to the individual variability of the bioavailability, the protein binding and short half-life of 30-150 minutes in order to avoid a possible overdose with an increased bleeding tendency or an underdose with an increased risk of thrombosis. In the clinical routine treatment with UFH this is most frequently monitored using the activated partial thromboplastin time (aPTT) and also by the thrombin clotting time (TCT) or activated clotting time (ACT). These assays are so-called global assays because they unspecifically reflect the thrombin-induced formation of a fibrin clot. The aPTT test which primarily determines the activity of the factors of the intrinsic system, is mainly sensitive to the inhibitory effects of heparin on thrombin. The aPTT test is sensitive for the heparin range of 0.1-0.7 U/ml but the normal range of aPTT as well as its therapeutic range depend very strongly on the reagent and the analyzer that is used. Another limiting factor is sample stability which often results in falsified results particularly after the blood sample has stood for too long. Further disadvantages of such global coagulation assays are among others the often complex experimental procedure which requires specially trained personnel to achieve reproducible results and the relatively high reagent consumption of these tests.
Monitoring is not absolutely necessary for normal patients when administering low-molecular-weight heparins due to their improved pharmacokinetics. However, it is recommended that the therapy is checked at the start of treatment and it is necessary especially in the case of patients with renal insufficiency due to their changed renal excretion and is recommended for patients with an extreme body weight, newborns, children and pregnant women or when they are used for several weeks or after fresh traumas or operations.
In clinical routine aPTT is used above all to monitor LMWH although this test has an only inadequate sensitivity for this anticoagulant and is moreover dependent to a very strong degree on the detection reagent that is used.
An FXa test is a suitable detection test for monitoring the effect of low-molecular-weight heparins.
Previous FXa tests are usually carried out as chromogenic tests or as clotting tests where the chromogenic tests measure factor Xa activity and the clotting test measures coagulation. Both test principles follow the same test procedure:                1. FXa+[heparin/antithrombin III]→[FXa/heparin/ATIII]complex+remaining FXa        2. a) remaining FXa cleaves a chromogenic residue from a FXa-specific substrate (chromogenic test)                    b) remaining FXa cleaves prothrombin to form thrombin (coagulation test via thrombin-induced fibrin cross-linking)                        
When the sample is added, factor Xa which is present in a defined amount in the test reagent binds with the heparin and antithrombin III contained in the sample and forms an inactivated complex with them. The remaining factor Xa cleaves either the chromogenic substrate or forms thrombin with the other coagulation factors contained in the sample and the thrombin cleaves fibrinogen to form fibrin (clot formation). More or less substrate is cleaved depending on the activity of factor Xa. The activity of factor Xa in turn depends on the amount of heparin contained in the sample. Whereas the chromogenic tests are specific for the FXa activity in the sample, coagulation tests do not exclusively measure the FXa activity but are nevertheless often more sensitive for LMWH than aPTT.
Several chromogenic tests but only a few coagulation tests (Heptest from the Sigma Company, ENOX test from the Pharmanetics Company) are commercially available. The various tests correlate only moderately with one another and with aPTT because they have different end points. The chromogenic tests yield activities but not clotting times. However, there is only a moderate correlation between the clotting time and factor Xa activity. Furthermore, these chromogenic tests require a separation of the plasma and they cannot be used directly in whole blood samples. As a result of the complicated sample preparation and process steps and the required devices, these methods of determination are time-consuming, labour intensive and require complicated apparatuses. Although the Heptest yields a clotting time as a result, it can, however, be very different depending on the different heparin sensitivity of the patient at the same heparin concentration. Furthermore, it is necessary to establish a heparin calibration curve from which the test result can then be read. Up to now only a single factor Xa test has been available as a dry chemistry test and thus also suitable for point-of-care instruments (ENOX test from the Pharmanetics Co.) which is carried out using a test card on the Rapid Point analyzer from Bayer AG. This test was specially developed for the use of Enoxaparin for percutaneous transluminal angioplasty and only distinguishes between Enoxaparin concentrations of >1 U/ml and <1 U/ml and is thus unsuitable for the routine monitoring of low-molecular-weight heparins especially because other fractionated heparins and unfractionated heparin interfere with the test.
WO 03/050298 describes the principle of this dry chemistry FXa test as follows: The sample to be examined which is preferably citrated whole blood, is admixed with a dry chemistry reagent which contains at least a factor Xa activator, preferably Russels viper venom and homogeneously dispersed magnetic particles. Factor X contained in the sample is converted into factor Xa by the factor Xa activator contained in the reagent and the factor Xa in turn results in the conversion of fibrinogen into fibrin via the prothrombin-thrombin conversion and thus the formation of the coagulation clot. This clot is detected by means of optical methods by observing the mobility of the magnetic particles in the reaction mixture which is caused by an external oscillating magnetic field. Hence this test principle is based on the formation of a fibrin clot which is only formed during the course of the detection reaction in a multistage reaction cascade triggered by factor Xa which involves further enzymes and cofactors in addition to factor Xa. Thus, for example the polymerization and cross-linking of fibrin requires the presence of calcium ions and activated factor XIIIa which is in turn formed from inactive factor XIII by a thrombin-dependent activation. Hence, the determination of factor Xa by means of the determination of a fibrin clot is also dependent on other essential factors and possible interfering effects. In addition to this indirect method of determination, the detection method described in WO 03/050298 requires a complex detection and evaluation system for the factor Xa determination. Thus, on the one hand, the test carrier on which the coagulation reaction takes place must have special devices which ensure a good and homogeneous mixing of the reagents and magnetic particles with the sample and, on the other hand, the evaluation system for determining the factor Xa activity must have devices for generating an oscillating magnetic field for example by means of a movable permanent magnet and optical systems for illuminating and photometrically detecting the movement of the magnetic particles.
WO 01/63271 (US2003/0164113) describes in general electrochemical sensors based on dry chemistry for determining blood coagulation or individual coagulation factors which have at least two electrodes on an inert carrier, as well as a dry reagent which contains a protease substrate which consists of a peptide residue that can be cleaved by a protease of the blood coagulation system and is amidically bound via its carboxyl end to substituted amines and in particular to a phenylenediamine residue. After the protease-induced cleavage, these substituted amines act as electron carriers of the 2nd type and can be used for the electrochemical determination of the protease activity. In addition to the so-called global tests such as aPTT, PT or ACT in which the clotting time is determined via the activity of the protease thrombin, WO 01/63271 (US2003/0164113) also describes tests that can be used to determine individual coagulation factors or their inhibitors. In this case WO 91/63271 teaches the use of substrates especially designed for the coagulation factor to be determined, the peptide part of which is specially adapted to the protease to be determined such that it can be specifically cleaved by this protease and the substituted amine as an electrochemically detectable particle specifically reflects the activity of this protease. When applied to a factor Xa test this would mean using a protease substrate which consists of a peptide residue that can be cleaved by factor Xa whose carboxyl end is amidically bound to substituted amines and especially to a phenylenediamine residue. To this extent the test principle is similar to that of chromogenic factor Xa tests in which an enzymatic cleavage product of a factor Xa-specific substrate is also used to determine factor Xa.
Accordingly, the inventors have identified a need in the art to provide methods for determining factor Xa inhibitors in blood samples which can be carried out simply even by persons that are not specially trained with low requirements with regard to time, apparatus or labor and which lead to reliable results in a short time. In particular, what is needed is a method for determining factor Xa inhibitors in blood samples which can be simply carried out and managed with the smallest possible number of process steps and required reagents and/or apparatuses thus enabling a rapid decentral analysis for example directly in intensive care units or hospital wards. It is desirable that the methods and devices for determining factor Xa inhibitors in blood samples also enable a determination directly in whole blood and thus do not require any complicated sample preparation steps. The methods and devices for determining factor Xa inhibitors in blood samples could satisfy the requirements for shelf-life and stability of reagents and enable a determination which is as accurate and specific as possible. In particular, a need exists for the above methods and devices for determining heparins, in particular fractionated heparins or low-molecular-weight heparins as well as direct factor Xa inhibitors in blood samples.